1. Field of the Invention
The present invention refers to the field of molecular biology and particularly to the possibility of finely controlling important gene functions for the somatic gene therapy. More specifically, the present invention provides a system for generating forms of some Rep proteins of Adeno-associated virus (AAV) dependant on steroid hormones. The invention also refers to the regulation of the AAV Rep proteins activity to direct the integration of recombinant DNA sequences within specific regions of human host-cell genomic DNA.
2. State of the Art
The integration of therapeutic genes into specific DNA sites of actively dividing cells and of non dividing cells, accompanied by prolonged expression, is the optimal strategy for somatic gene therapy.
The Adeno-associated virus (AAV) has the unique capacity of preferentially integrating its viral DNA into defined regions of the cellular genome, thus reducing the risk of insertional mutagenesis associated with other viruses, e.g. retroviruses that integrate in totally random positions.
The Adeno-associated virus (AAV) is a non-pathogenic small virus, with a single-stranded DNA, not capsized, belonging to the parvovirus family (Balaguxc3xa9 et al, 1997). AAV requires coinfection with a helper virus (adenovirus or herpes virus) or the host cells exposition to genotoxic agents (e.g. heat shock, hydroxyurea, UV light and X-rays) in order to undertake a productive infection. In the absence of a helper virus the AAV genome integrates into the chromosomal DNA to generate a latent infection. Analysis of flanking sequences of the provirus integrated genome, as the FISH analysis (in situ hybridisation with fluorescent probes) from latent infected cells, has revealed that the integration of the AAV genome is preferentially targeted to a specific site (called aavsl) localised in the q- terminus of the chromosome 19 (19q13-qter) (Kotin, R M, et al, 1990, PNAS USA, 87:2211-2215; Samulski. R J et al, 1991, EMBO J. 10:3941-3950]). The specificity of the integration refers to a spectrum of 60%-94% of the cases, depending on the cellular lines utilised and on the experimental conditions. This feature reduces the probability of insertional mutagenesis deriving from the random integration of the viral genome. Moreover, the absence of powerful transcription regulatory elements in the AAV genome makes it unlikely that the AAV integration in the aavsl site may be responsible of the transcription activation of endogenous chromosomal genes.
The integrated genome AAV can be rescued and replicated, if the cells containing an integrated provirus are superinfected with a helper virus like the Ad.
The AAV genome is a linear single-stranded DNA 4680 bp long, containing two sequences coding for proteins (ORFs=Open Reading Frames), three promoters (p5, p19 and p40), and an ITR sequence (Inverted Terminal Repeat sequence) of 145 bp, located at each end of the genome. The two ORFs code for non structural (Rep) and structural (Cap) proteins respectively (Kotin, R M, et al, 1990, PNAS USA, 87:2211-2215; Samulski. R J et al, 1991, EMBO J. 10:3941-3950).
The AAV ORF rep codes for four overlapping proteins. More specifically, the rep-coding ORF has two promoters located at map positions 5 (promoter 5, p5) and 19 (promoter 19, p19). The transcripts originated by each one of those two promoters share a common intron near to the 3xe2x80x2 terminus of the reading frame and to the polyadenilation site. The intron is utilised only in a subpopulation of the RNA messenger produced. Therefore, the ORF for rep generates four different mRNAs and the corresponding proteins: Rep78 and Rep68, expressed under control of p5 and Rep52 and Rep40, expressed under control of p19. Rep68 and Rep40 are coded by RNA transcripts that undergo a maturation process called xe2x80x9csplicingxe2x80x9d leading to the intron excision. Mutational analysis has shown the functional activities of the various Rep proteins.
Rep78 and Rep68 are multifunctional proteins that play a crucial role in the AAV replication. Rep78 is 621 amino acids long while Rep68 is 536 amino acids long: Rep78 and Rep68 differ only in their carboxy-terminal end, while the first 529 amino acids are identical in the two peptides. Rep78 and Rep68 share similar biochemical properties: both perform activities that are required for the AAV DNA replication, including the capacity of binding the RBS (Rep Binding Site) in the ITRs, and of cleaving site-specifically in a single strand manner the trs (terminal resolution site) present in the ITRs. Furthermore, Rep78 and Rep68 act as DNA-DNA and DNA-RNA helicases, have an ATP-ase activity and are capable of regulating positively or negatively both AAV promoters and heterologous promoters. Up to today evident functional differences between Rep78 and Rep68 have not been observed. Rep52 and Rep40, which do not show binding activity or endonucleasic DNA activity, are nevertheless important for the AAV infective cycle, as they promote the accumulation of single-stranded capsized genome of AAV.
AAV integration mechanisms during the non productive infection were not entirely clarified. Anyhow, it was clearly established that, beside undetermined cellular factors, two viral elements are required: the ITRs and the Rep78/68 (Carter, B J. in xe2x80x9cHandbook of Parvovirusesxe2x80x9d, ed. P. Tijsser, CRC Press, pp. 155-168, Samulski, R W096/36364). This conclusion is the direct consequence of many observations: firstly, recombining AAV vectors lacking of the rep and cap coding sequences do not specifically integrate into chromosome 19. Secondly, a RBS and a potential flanking trs were identified into the preferential genomic integration site aavsl, and it was proved that Rep68/78 can simultaneously bind the RBS present in ITRs and in aavsl, thus bridging the two DNA sequences. Moreover, utilising an ex vivo assay it was proved that a 33 bp sequence comprising the RBS and the trs in aavsl is the shortest sequence required in order to obtain the targeted integration of AAV into a DNA propagated as episome. Even more interesting is the observation that the two elements required for the AAV site-specific integration, function with a rather high efficiency even when utilised outside of the viral genome context. Actually, it was proved that plasmids bearing a rep expressing cassette can promote, when transfected in cells, the site-specific integration of a transgene flanked by ITR (integration cassette) contained either in the same or in a cotransfected plasmid.
Those experiments overall prove that it is possible to utilise the AAV integration mechanism in a different context from that of the AAV genome. This is of great relevance in the field of somatic gene therapy, because the primary limitation of AAV use for the somatic gene therapy is the low packaging limit of the AAV virion, that cannot exceed 4.5 Kb. As a recombining vector AAV able to integrate specifically in the human chromosome 19 ought to contain Rep78 and/or 68 cDNA (about 2000 nt) as well, the wider DNA sequence (e.g. transcription regulating regions 5xe2x80x2 and 3xe2x80x2+transgene) transductable with the AAV vector could not be longer than 2-2.5 Kb.
These dimensional limitations could be overcome either by transducing the ITR-flanked transgene and the rep expression cassette with a non-viral system, or by the introduction of those elements into a viral vector with a wider cloning capacity (e.g. adenovirus, baculovirus, herpes virus, etc.) (as disclosed in the Italian patent application RM97A000200, priority date Apr. 8, 1997). Whatever the selected transduction system be, a tight regulation of the functioning Rep78/68 protein(s) is necessary.
In nonviral transduction systems, it would be necessary to have Rep proteins functioning only for the time required to obtain the integration, in order to avoid any undesired influence on the cell physiology. This is especially true in light of the observation that Rep exerts a cytotoxic-cytostatic effect on cell cultures (Yang et al, 1994). Moreover, a tight control of the Rep activity on target cells might be required as well, in order to eliminate any possible Rep-dependant recombination, subsequent to the, original integration event.
In the construction of hybrid viral vectors such as adenovirus vectors (Ad)/AAV, i.e. Ad vectors comprising the rep68/78 coding sequence, the AAV ITR and a transgene (with its regulating sequence) among the AAV ITRs, the regulation of Rep activity is required for at least two reasons. Firstly, it is known that Rep can suppress the growth of many viruses, like SV40, HIV, herpesvirus, and Adenovirus. In this last case, particularly, the Rep capacity of inhibiting Ad growth was exhaustively characterised: therefore, in order to generate hybrid Ad/AAV viruses it is necessary to specifically restrain Rep activity on the cell line wherein the adenoviral vector packaging takes place (293 cells derivated from human embryo kidney), otherwise virus yield would be significantly lowered. Whatever be the hybrid virus constructed, in the cell line wherein the packaging takes place Rep activity must be kept low, also to maintain the integrity of the vector genome. As a matter of fact, if it were active during the virus growth, Rep by interaction with the AAV ITRs could promote the transgene excision (and possibly its replication, as it is known that the AAV ITRs may function as replication origin of the eucaryotic cells) off the hybrid vector genome, therefore generating non homogeneous viral populations. On the contrary, obviously, Rep78 and/or Rep68 must be active inside target cells for the time needed to promote the site-specific integration of the desired transgene on the aavsl site.
All these considerations point out that the generation of Rep proteins 78/68, depending for their activity on a ligand added from the outside, would be extremely useful in the field of the somatic gene therapy.
In particular, as the AAV virus most commonly used in gene therapy is the AAV virus of type-2 (AAV-2), applying such a generation of dependant Rep proteins on this specific AAV type virus, would be even more extremely useful.
The subject matter of the present invention are the hormone-dependant forms of the Rep proteins 78 and Rep 68 of the Adeno-associated virus (AAV), obtained by the fusion of their specific mutants with the hormone binding domain (HBD) of steroid hormone receptors, and the DNA sequences coding for them. These mutant forms in fact enable to obtain a system for the regulation at the post-translational level of the activity of Adeno-associated virus (AAV) Rep proteins by the deletion of the sequence responsible of their localisation in the nucleus (nuclear localisation signal-NLS) and the fusion of the truncated protein with the HBD.
A main discovery which the present invention is based on, consists in the impossibility of regulating the activity of the native proteins Rep 68 and 78. Actually, both Rep 68 and Rep 78 share the same NLS. The whole wild type proteins Rep 68 and Rep 78, in fact, are not regulated by fusion with HBD for steroid hormone, as to both intracellular localisation and the capacity of promoting site-specific integration, because of the presence of this co-shared region. As a consequence, was developed and will be disclosed hereinafter a method object of the present invention by which the Rep protein, truncated in the carboxy-terminal region so as to present a partial or total NLS deletion, becomes subjected to regulation, with the fusion with HBD for steroid hormone.
The partial or total inactivation of the NLS localised in a region embracing the amino acids 480-520 of the Rep proteins 68 or Rep78 primary sequence, can be obtained by different mutations, the most effective being the one consisting in the deletion of the whole domain or of parts of it.
More specifically, the capability of the Rep proteins 68 or 78, deleted from amino acid 505 to amino acid 520, of promoting site-specific integration, can be regulated by fusion with HBD for steroid hormone.
Preferably, the propriety of being regulated by fusion with HBD is obtained by the deletion of the amino acids 492-520, and more preferably by the deletion of the amino acids 485-520.
The regulation of the capacity to promote the site-specific integration of the mutant rep peptides, could be reached by the fusion both at the amino- end and at the carboxy-terminal end of the rep mutants with the HDB for steroid hormones, preferably the regulation of activity of the mutant rep peptides is reached by fusion of the carboxy-terminal end of the mutant rep peptides with the HBD for steroid hormones.
Preferably the HBD utilised is that of the receptor for human progesterone comprising amino acids 640-933 (hPR-HBD)and even more preferably one of its mutants, derivated by the deletion of the C-terminal 42 amino acids, therefore consisting of the amino acids 640-891 (PR891).
In the light of the aforementioned and of what shall be exposed hereinafter, subject matter of the present invention are muteins of the Rep proteins 68 or Rep 78 of an Adeno-associated virus, comprising at least one mutation in a region comprised from residue 480 to residue 520, said mutation being capable of partially or totally inactivating the nuclear localisation signal, being optionally present the wild type Rep 68 or Rep 78 sequence from residue 521 to the carboxy-terminus, said mutein being fused with the binding domain for a steroid hormone.
Particularly, the cases are considered wherein said mutein derives from the wild type protein by the deletion of at least one amino acid of the region comprised from amino acid 480 to amino acid 520. Furthermore, cases wherein the deletion comprises residues 485-520, 492-520 and 505-520, and cases wherein, there existing one of the aforementioned deletions, the region from amino acid 521 to the carboxy-terminus of the sequence of wild type Rep 68 or Rep 78 is absent, are of particular relevance.
A further subject of the present invention are the cases wherein the binding domain for the steroid hormone is situated at the carboxy-terminal end of the Rep 68 or Rep 78 mutein, and is selected from the group comprising the hormone binding domain of the progestinic receptors, the hormone binding domain of the estrogen receptors, the hormone binding domain of the glucocorticoid receptors, the hormone binding domain of the mineralcorticoid receptors, and muteins derivated thereof, particularly the ones wherein said receptors and said hormones are of mammal origin, and particularly that wherein they are of human origin.
Further particular cases are the muteins wherein: the binding domain of the receptor for the progesterone consists in the sequence of amino acids from the residue 640 to the residue 933, in particular said domain is mutated for deletion of the 42 C-terminal amino acids, and consists of the amino acids 640-891; the binding domain for a steroid hormone is situated at the carboxy-terminus of the Rep 68 or Rep 78 mutein.
The present invention further refers to a mutein of the Rep proteins 68 or 78 of an Adeno-associated virus, comprising at least one mutation in a region comprising amino acids from residue 485 to residue 520, and those mutations wherein, there being one of the aforementioned deletions, the region from amino acids 521 to the carboxy-terminal end of the sequence from wild type Rep 68 or Rep 78 is absent.
Moreover, of particular relevance is the case wherein the mutein comprises at least one mutation in a region from residue 480 to residue 520, being optionally present the sequence of wild type Rep 68 or Rep 78 from residue 521 to the carboxy-terminal end.
More particularly relevant is the case wherein the Adeno-associated virus is the Adeno-associated virus of type 2.
The present invention also refers to the DNA sequences, in particular cDNA, coding for the aforementioned muteins, and vectors comprising them, viral or plasmidic, with particular reference to vectors comprising sequences described in the text or in the examples; as Repxcex94N-P, Rep1xcex94N/Pn and Rep1xcex94N/P.
Moreover, a further subject of the present invention is a method for regulating the intracellular activity of the Rep 68 or Rep 78 polypeptides of an Adeno-associated virus, essentially comprising a combination of the following steps:
a) introducing in a cell the DNA sequence coding for at least a Rep 68 or 78 protein as previously described, having the nuclear localisation signal (NLS) partially or totally inactive, said DNA sequence being operatively linked to a sequence coding for a steroid HDB, or a derivative thereof;
b) adding a steroid hormone or analogs of the steroid hormone capable to bind said binding domain.
Particular relevance has the cases wherein said DNA sequence codes for the muteins as previously described and is inserted into the cell by viral vector infection, in particular the vectors previously described, or with a transfer technique of the selected DNA from the group comprising electroporing, DEAE-dextran transfection, calcium-phosphate transfection, DNA gun, and liposome-mediated genic transduction.
A further relevant case is that wherein the binding domain of the steps a) and b) are those previously described.
A similar method for regulating the intracellular activity of polypeptides Rep 68 or Rep 78 of an Adeno-associated virus, comprising essentially a combination of the following steps:
A) introducing into a cell a mutein of a Rep 68 or 78 protein having a nuclear localisation signal (NLS) partially or totally inactive, fused to a sequence coding for a binding domain for a steroid hormone or a derivative thereof;
B) adding a steroid hormone or a steroid hormone analogous substance capable to bind said binding domain.
In this method any one of the muteins described herein may be used.
Particularly, the case is considered wherein the regulating method of the intracellular activity of the polypeptides Rep 68 or Rep 78 is applied to eucaryotic cells, preferably mammalian, and most preferably human. A particular case illustrated is the use of 293 cells.
In a preferred embodiment the mutein can be inserted into the cell by liposomes.